Screening and ldentification of Antagonistic Bacteria against Soil-borne Diseases of Tomato

Xuejie LlANG，Tingting ZHANG，Junqing QlAO，Yan DU，Youzhou LlU*.Institute of Plant Protection，Jiangsu Academy of Agricultural Sciences，Nanjing 004，China；.Southern College，Nanjing Forestry University，Nanjing 0037，China

Screening and ldentification of Antagonistic Bacteria against Soil-borne Diseases of Tomato

Xuejie LlANG1，Tingting ZHANG2，Junqing QlAO1，Yan DU1，Youzhou LlU1*
1.Institute of Plant Protection，Jiangsu Academy of Agricultural Sciences，Nanjing 210014，China；
2.Southern College，Nanjing Forestry University，Nanjing 210037，China

［Objective］This study was conducted to screen resources for biological control of soil-borne diseases of tomato.［Method］Antagonistic bacteria against Ralstonia solanacearum and Fusarium oxysporum were isolated and purified from soil samples by dilution plating and confronting incubation on PDA plates，then the antibacterial spectrum and metabolic secretions of the bacterial isolates were measured，and their species were identified by establishing phylogenetic trees with Neighbor-Joining method.［Result］From the 60 soil samples，10 of 59 antagonistic bacteria isolates against R.solanacearum showed inhibition zone diameter＞25 mm，and 4 of 30 strains against F.oxysporum exhibited inhibition rate＞30%.The bacteriostatic substances and antibacterial spectrum analysis on above 14 isolates revealed that four strains N23-2，N58-2，NF59-3 and NF61-1 had good antagonism against pathogenic bacteria and fungi；12 strains produced cellulose，11 strains produced proteases，and 6 strains produced siderophores.The molecular identification experiments indicated that four strains were members of Paenibacillus，three strains of Streptomyces，three strains of Pseudomonas strains，and four strains of Bacillus strains.

Ralstonia solanacearum；Fusarium oxysporum；Antagonistic bacteria；Screening；Identification

T omato as a nutritious fruit is widely grown throughout the world.In recent years，tomato planting area and cropping index gradually grew as a result of industrial structure adjustment，while the incidence of soil-borne diseases on tomato also increased year by year，causing more and more loss to this industry.Especially，tomato bacterial wilt which is caused by Ralstonia solanacearum and tomato blight which is caused by Fusarium oxysporum are two important soil-borne diseases.The incidence rate of the former ranges from 10%to 15%generally，up to 80%-98%in some severe attacks of R.solanacearum［1］；tomato blight can cause plant wilt and death in large scale，and its incidence rate is 20%to 30%in general，up to 80%in some sever attacks［2］.

Currently，agrochemicals are still the dominant approach for the control of tomato bacterial wilt and blight in production，which cause serious pollution to the ecological environment and also increase residues of toxic chemicals in agricultural products.Biological control has recently attracted much attention because it is safe for human，animal and environment， without causing drug resistance to pathogens. So far，many effective bacteria for biocontrol have been reported，such as Bacillus spp.［3-4］，Pseudomonas spp.［5-6］，and Streptomyces spp.［7-8］.

In the present study，we isolated several bacterial strains those had antagonistic activity against R.solanacearum and F.oxysporum from the soil samples collected around vegetable roots in Jiangsu，Jiangxi and Zhejiang and other provinces，and then analyzed their antibacterial spectrum and metabolic secretions，identi-fied their species，with an attempt to provide more microbial resources for biological control of soil-borne diseases in tomato.

Materials and Methods

Materials

Pathogens Four pathogenic fungi Fusarium oxysporum， Rhizoctonia solani，Botrytis cinerea，Sclerotinia sclerotiorum and three pathogenic bacteria Ralstonia solanacearum，Xanthomonas oryzae，Erwinia amylovora were provided by the Institute of Plant Protection，Jiangsu Academy of Agricultural Sciences.

Media PDA medium was prepared for the cultivation of fungi，and LB medium for the culture of pathogenic bacteria and antagonistic bacteria［9］. Metabolic secretions of the antagonistic bacteria were detected on four selective media for protease［10］，chitinase［11］，cellulase［12］and siderophore assay［13］.

Methods

Isolation and screening of antagonistic bacteria A total of 60 soil samples were collected around vegetable roots，from which antagonistic bacteria were isolated by dilution plating［9］.In detail，100 μl of 1∶1 000，1∶10 000 and 1∶100 000 dilutions of every soil sample were coated on solid LB and PDA plates，cultivated in an incubator at 28℃ for 1-2 d.All the pathogenic bacteria，pathogenic fungi and antagonistic bacteria were isolated，purified and cultured in LB and PDA media separately.

Activity measurement of the antagonistic bacteria

Antagonistic bacteria against R.solanacearum R.solanacearum was activated and inoculated into 20 ml of LB liquid medium，cultured at 28℃and 150 r/min for 24 h，and finally diluted with sterile water to a concentration of 106cfu/ml.Then the dilution was inoculated by instant spraying into the LB medium plates prepared in above steps，cultured at 28℃for 24 h. The single colonies had obvious inhibition zone were further isolated and purified by streaking inoculation.The selected strains antagonistic against R.solanacearum were inoculated into LB liquid medium，cultured by shaking overnight.After that，5 μl of the medium was inoculated at the central point of blank LB plates，cultured at 28℃for 48 h before the instant spraying of R. solanacearum.After another 24 h of culture at 28℃，the inhibition zone diameter was measured.Three replications were set for each treatment.

Antagonistic bacteria against F.oxysporum F.oxysporum was cultured on PDA plates for 7 d before its spores were washed and diluted to a concentration of 106 spores/ml with sterile water.The spore solution was sprayed into the PDA plats prepared in“Isolation and screening of antagonistic bacteria”，cultured at 28℃for 24 h. The single colonies had obvious inhibition zone were further isolated and purified by streaking inoculation.The mycelial disc of F.oxysporum was inoculated at one side of blank PDA plates （30 mm away from the plate edge），and 5 μl of antagonistic bacteria against F.oxysporum was inoculated into the other side of the PDA plates，with three replications for each treatments.The antagonistic bacterial solution was replaced by sterile water in control group.Then，the PDA plates were cultured at 28℃.The mycelial growth diameter of F.oxysporum in antagonistic bacteria plates was measured when F.oxysporum mycelia fully covered the plates of control.Inhibition rate of the antagonistic bacteria against F.oxysporum were calculated according to the formula as follows:Inhibition rate=（F.oxysporum mycelial growth diameter of control-F.oxysporum mycelial growth diameter in antagonistic bacteria plate）/ F.oxysporum mycelial growth diameter of control×100%.

Determination of antibacterial spectrum of the antagonistic bacteria Antagonistic activities of the bacteria against F.oxysporum，R.solani and E.amylovora were measured as described in“Antagonistic bacteria against R.solanacearum”.And the antagonistic activities of the bacteria against B.cinerea，S.sclerotiorum，R.solanacearum and X.oryzae pv. oryzicola were measured as described in"Antagonistic bacteria against F.oxysporum".

Determination of metabolic secretions of the antagonistic bacteria

The purified antagonistic bacteria isolates were activated，and single colonies were inoculated into 5 ml of liquid medium and cultured at 28℃and 150 r/min for 24 h.5 μl of liquid medium of each antagonistic bacterium were inoculated at the center of plates of four selective media，cultured at 28℃for 3-7 d before the assay of protease［10］，chitinase［11］and cellulase［12］and 7-10 d before the assay of siderophore［13］.Three replications were prepared for each treatment.Positive reactions of chitinase，protease and cellulase would show transparent circles around the colonies，and positive reaction of siderophore would exhibit distinct yellow halos.

Species identification of the antagonistic bacteria The bacterial DNA was extracted by conventional methods，detected through electrophoresis on 1%agarose gel for purity and concentration.Then the extracted genomic DNA was used as a template to amplify 16S rRNA sequences，using universal bacterial primers:BSF（27 F）:AGAGTTTGATCCTGGCTCAG and BSR （1 492 R）:TACGGYTACCTTGTTACGACTT.The PCR reaction was started with an initial denaturation at 95℃for 4 min，followed by 25 cycles of 40 s at 94℃，45 s at 55℃，and 1 min at 72℃，and a final extension at 72℃ for 5 min.The PCR products were preliminarily verified by 1%agarose gel electrophoresis and then sequenced by Sangon Biotech（Shanghai）Co.，Ltd.By submitting sequences to Genbank their accession numbers were obtained.Finally，phylogenetic trees were established using Clustal-x and MEGA 4 software with Neighbor-Joining method based on their 16S rRNA sequences.

Results and Analysis

Inhibitory effects of antagonistic bacteria against R.solanacearum and F.oxysporum

A total of 59 antagonistic bacteria strains against R.solanacearum were finally isolated from the 60 soil samples.Ten of them had inhibition zone diameter＞25 mm；particularly，three strains N23-2，N24-2and N56-1 showed inhibition zone diameter of 38.5，39.2 and 40.3 mm，all over 35 mm（Table 1 and Fig.1）.In addition，30 antagonistic bacteria strains againstF.oxysporum were isolated，four of which exhibited inhibition rate over 30%；particularly，two stains NF59-3 and NF61-1 had inhibition rate of 47.75%and 46.85% ，respectively，both greater than 45%（Table 2）.

Table 1 Inhibition of antagonistic bacteria against R.solanacearum mm

Table 2 Inhibition of antagonistic bacteria against F.oxysporum

Antibacterial spectrum of antagonistic bacteria

Antagonistic activities of the 14 bacterial isolates in Table 1 and Table 2 against the three plant pathogenic bacteria R.solanacearum，X.oryzae pv.oryzicola，E.amylovora and the four plant path-ogenic fungi F.oxysporum，R.solani，B.cinerea and S.sclerotiorum were detected（Table 3 and Table 4）.Among them，two strains N23-2 and N58-2 which had obvious antagonistic effects on R.solanacearum，also inhibited the mycelial growth of F.oxysporum，with the inhibition rate of 43.25%and 39.10%（Fig.2 and Fig.3）.Strains NF59-3 and NF61-1 which had antagonistic effects on F.oxysporum，also inhibited the growth of R.solana-cearum，with the inhibition zone diameter of 32.5 and 33.3 mm.In summary，the four strains N23-2，N58-2，NF59-3 and NF61-1 had obvious antagonistic effects against both R.solanacearum and F.oxysporum.Moreover，the four strains also inhibited the growth of two pathogenic bacteria and four pathogens fungi tested，which proved their broad-spectrum antimicrobial activity.

Strains N22-1 and N32-5 had obvious inhibitory effects on R.solanacearum，but no significant inhibitory effects on other pathogenic bacteria and fungi.Strain NF21-1 had obvious inhibitory effect on only F.oxysporum，with inhibition rate of 34.60%，and no or weak inhibitory effect on other pathogenic bacteria and fungi.In contrast，strain N24-2 had obvious inhibitory effects on all pathogenic bacteria and most pathogenic fungi tested，but its inhibition rate against F.oxysporum was only 14.05%.Strain N56-1 inhibited the growth of all the pathogenic bacteria and fungi tested except X.oryzae pv.oryzicola.

Strains N26-1 and N77-3 had strong inhibitory effects on all three pathogenic bacteria，but different effects on the four pathogenic fungi. Strain N26-1 had very weak inhibitory effect on R.solani and the inhibition rate was only 3.84%，but strong in-hibitory effects on other three pathogenic fungi.On the contrary，strain N77-3 had strong inhibitory effect on R.solani with inhibtion rate of 42.27%，but weak inhibitory effects on other three pathogenic fungi.

Strains N62-2 and N74-3 had a clear zones’of inhibition against all three pathogenic bacteria，but their inhibitory effects against pathogenic fungi were very poor.Stain NF22-1 did not inhibit the growth of R.solanacearum，and its diameters of inhibition zone diameter against X.oryzae pv.oryzicola and E.amylovora were only 14.2 and 13.3 mm.However，it had specific inhibitory effects on the four pathogenic fungi，and formed obvious inhibition zone around the colonies.

Activity of metabolic secretions of the antagonistic bacteria

Twelve of the 14 antagonistic bacteria strains secreted cellulase while two strains N62-2 and NF21-1 did not.Five strains N23-2，N24-2，N26-1，N74-3 NF59-3 and NF61-1 secreted siderophore.Eleven strains secreted protease while three strains N56-1，NF21-1 and NF22-1 did not. All the 14 antagonistic bacteria did not produce chitinase.

Species identification of the antagonistic bacteria

Three phylogenetic trees were finally established by comparing the 16S rRNA sequences of strains N22-1，NF21-1 and NF22-1 with the model strains of genus Pseudomonas，the 16S rRNA sequences of strains N56-1，N62-2 and N74-3 with the model strains of genus Streptomyces，and the 16S rRNA sequences of other eight strains with the model stains of genus Bacillus in NCBI（Fig.4，F(xiàn)ig.5 and Fig.6）.Among the 14 antagonistic bacteria，three stains belonged to genus Streptomyces，three to genus Pseudomonas，four to genus Paenibacillus and four to genus Bacillus（Table 5）.

Table 3 Inhibition efficiency of 14 antagonistic bacteria against the growth of three plant pathogenic bacteria mm

Table 4 Inhibition efficiency of 14 antagonistic bacteria against the growth of four plant pathogenic fungi

Discussion

The four strains N23-2，N58-2，N59-3 and NF61-1 we isolated in this study exhibited strong antagonistic activities against R.solanacearum and F.oxysporum，but also inhibited the growth of many other pathogenic bacteria and fungi，which proved their broad-spectrum antibacterial activity. Their biocontrol efficiency will be de-tected in pot and field experiments in future to provide theoretical basis for the development of biological pesticides for the control of soil-borne diseases in tomato.

Paenibacillus polymyxa not only plays a role in disease prevention［14］，but also promotes plant growth［15］，besides，it is safe to human，animal and environment.Therefore，it was ranked one of the applied microorganisms in commerce by the US.Environmental Protection Agency（EPA）［16］.P.polymyxa can produce peptides，proteins，nucleosides and other antibacterial substances which can inhibit the growth of various pathogenic bacteria and fungi.Lipopeptides secreted by P.polymyxa such as polymyxins can act on cell membrane of bacteria and destroy biological activity of the membrane，thereby causing the death of bacteria［17］.Polymyxin has specific inhibitory effects on G-bacteria as their cell wall contains more phospholipids. P.polymyxa JB05-01-1 which was isolated by Naghmouchi et al.［18］from livestock feed produced a broadspectrum antimicrobial protein，which inhibited the growth G+bacteria.Chen et al.［19］found that antibacterial substances produced by P.polymyxa CP7 that caused irregular changes and wilt to Peronophythora litchi mycelia，destroyed its spore structure and inhibited spore germination.Four of the antagonistic bacteria strains N23-2，N32-5，NF59-3 and NF61-1 in our study were classified to genus Paenibacillus. Among them，strains N23-2，NF59-3 and NF61-1 showed broad-spectrum antimicrobial activity as they had obvious inhibitory effects on the tested pathogenic bacteria and fungi.However，strain N32-5 had strong inhibitory effects on only F.oxysporum，but weak inhibitory effects on other pathogenic bacteria and fungi，which was inconsistent with previous studies. So，further studies should be performed to analyze the activity of its antibacterial substances.In addition，we found that the antagonistic bacteria even in a genus had different inhibitory effects on pathogens.Similar phenomenon was found in all the four genera.

Strains N22-1，NF21-1 and NF22-1 were identified as Pseudomonas spp.However，strains N22-1 exhibited obvious inhibitory effects only on R.solanacearum，and NF21-1 only on F.oxysporum，while NF22-1 had weak inhibitory effects on pathogenic bacteria，but strong inhibitory effects on pathogenic fungi.Such differences in inhibitory effects on pathogenic bacteria and fungi among the three Pseudomonas spp.strains indicatedthat they could produce different antimicrobial substances.

Table 5 16s rRNA sequence homology analysis of 14 antagonistic bacteria

Bacillus spp.N26-1 and N77-3 also exhibited different inhibitory effects on the four pathogenic fungi.In detail，N26-1 had little inhibitory effects on R.solani，but strong inhibitory effects on other pathogenic fungi，while N77-3 had strong inhibitory effects on R.solani，but weak inhibitory effects on other pathogenic fungi.Ge et al.［20］also reported that Brevibacillus sp.JK-2 had strong and stable inhibitory activity on F.oxysporum，and it also exhibited obvious inhibitory effects on other five plant pathogenic fungi，Verticilliumalbo atrum，V.dahlia，V fungicola，Colletotrichum gloeosporioides and Monilia fructicola Poll，but no inhibitory effects on B.cinerea. Therefore，the secretions of straine N26-1 and N77-3 will be purifieds and analyzed to find out their difference in the future work.

Liu et al.［21-22］found that Streptomyces spp.inhibited the mycelial growth and spore germination of F.oxysporum f.sp.cubense and F.oxysporum f.sp.cucumebrium. The study of Wang et al.［23］revealed that the metabolites of Streptomyce sp.R15significantly inhibited the growth of several pathogenic fungi such as F.oxysporum f.sp.vasinfectum，Phytophthora capsici Leonian and Colletotrichum micotianae Averna. In this study，we found that Streptomyces sp.N56-1 inhibited the growth of all the pathogenic bacteria and fungi tested except X.oryzae.However，pathogenic bacteria and fungi better. But Streptomyces spp.N62-2 and N74-3 had inhibitory effects on all the three pathogenic bacteria，but their inhibitory effects against pathogenic fungi were very poor，which was not consistent with previous studies，suggesting that their secretions might had strong inhibitory effects on pathogenic bacteria but weak inhibitory effects on pathogenic fungi.

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Responsible editor:Qingqing YIN

Responsible proofreader:Xiaoyan WU

（Continued from page 529）

Optimum temperature for pathogen growth

This strain was inoculated into a PDA plate and cultured at temperatures of 10，15，20，25，30，35 and 40℃for 5 d，with relative humility of 75%，the daily growth curve of mycelium was shown in Fig.4.

As shown in Fig.4，F(xiàn).venfricosum could grow in a wide temperature range from 10 to 40℃ ，and the mycelium grew faster from 15 to 35℃. The daily growth of mycelium length was largest at 30℃，up to 38.5 mm，so 30℃was the optimum temperature for the growth of F.venfricosum.

Fungicides for pathogen control in laboratory

The strain was inoculated into the media supplemented with different toxics，cultured at 25 to 30℃for 5 d. The results were shown in Table 1.

As shown in Table 1，all the tested fungicides in the media inhibited the growth of this strain to some degree，their effects in a descending order were as follows:carbendazol，thiophanate-methyl，chlorothalonil，thiram and mancozeb.

Conclusions and Discussion

Rubber collar rot caused by F.venfricosum in this study was the first report at home and abroad.The only known Fusarium sp.causing rubber disease is F.tumidum which can cause rubber tree dieback and canker［2］.

Fusarium is one of the most economically valuable fungus and most difficult to be identified.Fusarium spp. are mesophiles，most distributed in tropical and sub-tropical zone.F.venfricosum grew well at temperatures from 10 to 40℃，better from 15 to 35℃，fastest at 30℃，and their growth was inhibited above 40℃or below 10℃.All the results prove that F.venfricosum is a mesophile.

Fusarium has many species，most of which are common and important plant pathogenic fungi，but few reports on plant disease caused by F.venfricosum.Loss percentage in some sugarbeet plant caused by F.venfricosum reached to 10%to 15%［2］.This fungi can be found in must tobaccos of the Federal Republic of Yugoslavia［2］.The pathogenicity of F.venfricosum was not strong to wheat according to Sang［4］.Li et al.［5］also found F.venfricosum in potato plant.

Table 1 Chemical control of F.venfricosum mm

The water accumulation in wet season was the reason causing this rubber collar rot in Xishuangbanna. There were no drainage ditches along the affected rubber trees，where water flew down and accumulated，resulting in the occurrence of this disease on rubber trees.Therefore，in the process of natural rubber cultivation，regulations and requirements for natural rubber cultivation should be performed strictly to avoid the occurrence of this disease，and to make sure the growth and tapping of rubber tree in a normal way.

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Responsible editor:Qingqing YIN

Responsible proofreader:Xiaoyan WU

番茄土傳病害拮抗菌的篩選和鑒定

梁雪杰1，張婷婷2，喬俊卿1，杜 艷1，劉郵洲1*
（1.江蘇省農(nóng)業(yè)科學(xué)院植物保護(hù)研究所，江蘇南京 210014；2.南京林業(yè)大學(xué)南方學(xué)院，江蘇 南京 210037）

［目的］篩選出對(duì)番茄土傳病有防治效果的生防菌。［方法］該研究以番茄青枯病菌（Ralstonia solanacearum）和番茄枯萎病菌（Fusarium oxysporum）為靶標(biāo)菌，利用梯度稀釋涂板法和平板對(duì)峙生長(zhǎng)法從土樣中分離、篩選出分別對(duì)這2種病原菌具有較強(qiáng)抑制作用的拮抗細(xì)菌；并對(duì)其抑菌譜及代謝分泌物進(jìn)行測(cè)定，運(yùn)用Neighbor-Joining方法構(gòu)建系統(tǒng)發(fā)育樹，對(duì)拮抗細(xì)菌進(jìn)行種屬鑒定。［結(jié)果］從60份土樣中分離出對(duì)番茄青枯病菌有拮抗作用的細(xì)菌59株，其中抑菌圈直徑 ＞25 mm的有10株；對(duì)番茄枯萎病菌有拮抗作用的細(xì)菌30株，其中抑制率 ＞30%的有4株。上述14株菌株中，N23-2、N58-2、NF59-3、NF61-1對(duì)多種病原細(xì)菌和病原真菌皆具有較好的拮抗作用；產(chǎn)纖維素酶的菌株有12株，產(chǎn)蛋白酶的菌株有11株，產(chǎn)嗜鐵素的菌株有6株。種屬鑒定結(jié)果表明：有4株屬多粘芽孢桿菌（Paenibacillus），3株屬鏈霉菌（Streptomyces），3株屬假單胞菌（Pseudomonas），4株屬芽孢桿菌（Bacillus）。 ［結(jié)論］該研究結(jié)果為番茄土傳病害的生物防治提供更多的微生物資源。

番茄青枯病菌；番茄枯萎病菌；拮抗細(xì)菌；篩選；鑒定

江蘇省農(nóng)業(yè)科技自主創(chuàng)新資金項(xiàng)目［CX（14）2058］。

梁雪杰（1988-），女，山東濟(jì)寧人，碩士，研究方向：植物病害生物防治，E-mail：L_xuejie@163.com。*通訊作者。

2014-11-20

2015-00-00

Supported by Fund for Independent Innovation of Agricultural Science Technology［CX（14）2058］。

.E-mail:shitouren88888@163.com

November 22，2014Accepted:000